Evidence for membrane cholesterol as the common binding site for cereolysin, streptolysin O and saponin

Addition of alfalfa saponin to osmotically prepared rabbit erythrocyte membranes resulted in formation of pits or holes as revealed by negative staining followed by electron microscopic examination. The effect on the membranes was quantitatively related to the saponin concentration. Alfalfa saponin also caused disruption of liposomes made of α-lecithin and cholesterol but not of liposomes made of α-lecithin alone. Pretreatment of osmotically prepared erythrocyte membranes with alfalfa saponin prevented ferritin labeling of the membranes by cereolysin-ferritin conjugate, and the degree of inhibition was a function of the concentration of saponin used in pretreatment. Liposomes composed of both lecithin and cholesterol inhibited the hemolytic activity of cereolysin and saponin whereas liposomes containing lecithin only did not. Incubation of cereolysin or streptolysin O with untreated erythrocyte membranes caused complete inhibition of hemolytic activity whereas incubation with erythrocyte membranes pretreated with cereolysin, streptolysin O, filipin or alfalfa saponin did not. Incubation of cereolysin or streptolysin O with erythrocyte membranes pretreated with soybean saponin resulted in complete inhibition of hemolysis. The results provide strong support for the idea that alfalfa saponin, filipin, cereolysin and streptolysin O share a common membrane binding site and the binding site is cholesterol.